(Macaca fuscata yakui) in Yakushima
Transcript of (Macaca fuscata yakui) in Yakushima
Non-Invasive Technique of Assessing the
Ecology of the Japanese Macaque
(Macaca fuscata yakui) in Yakushima:
Focusing on their Feeding of Fig Fruits
September 2012
Monkey Team
Lecturers: Dr. Sugiura, Hideki
Dr. Inoue, Eiji
Members: 1. Elisa Panjang (Malaysia)
2. Furukawa, Saori (Japan)
3. Nachiketh S. Ramamurthy (India)
4. Nishant M. Srinivasaiah (India)
5. Wakamori, Hikaru (Japan)
INTRODUCTION
A training course, aimed at assessing the ecology of Japanese macaque using
non-invasive technique was held from 8th to 13th Sepetember at Yakushima and from
18th to 21st at Wildlife Research Centre at Kyoto. This is a report culminating from
this exercise.
Non-invasive DNA sampling technique is considered a key for studying
animals without disturbing or influencing their behavior in the wild (Broquet et. al
2007). Therefore we collected the feces of Japanese macaque in Yakushima (Macaca
fuscata yakui) to extract DNA.
‘Yakushima’ is an island located in the south-southwest of Cape Sata on the
Osumi Peninsula of Kagoshima Prefecture, Japan. It has about 500 km² in area and is
circular in shape. It has its unique species in fauna and flora.
Wildlife Research Centre is one of the research centres in Division of
Biological Science, Graduate School of Science of Kyoto University.
OBJECTIVES
The first objective was to identify the sex of the macaque. Sex identification is
important to assess the composition of macaque groups. We can get this information by
observation, however, this time we did both, observational and DNA analysis; to
validate our data.
The second was to identify the species of the ficus fruits the macaques ate.
Yakushima macaque is known to predominantly feed on ficus fruits species (Hill 1997,
Otani et. al 2000). This study may help us to assess the preference of certain ficus fruit
species over the others by macaques during this season.
METHODOLOGY
In order to understand and elucidate the ecology of macaques using a
non-invasive DNA technique, feces was collected from macaques in the wild with
minimal or no disturbance to their lives. Fecal DNA includes the DNA of animals,
plants which the macaques digested, bacteria, virus, and parasites, which can provide
valuable information on the ecology of wild animals. For this study, emphasis was laid
towards identification of the sex of the macaque and the ficus species they ate.
The following steps were employed to identify the sex of the macaque and the
species of ficus they fed on both In-situ and Ex-situ;
1) DNA collection from feces for sex identification
a. Fecal samples, including epithelial cells from nine fecal samples and one blood
sample of macaque, belonging to three groups, were collected and stored in lysis
buffer.
b. Eight fecal samples’ sexes were identified by direct observation before
collection.
We preserved the surface of feces or blood in 2ml tube with lyses buffer.
2) Seed collection from feces for species identification
Seeds, especially of ficus trees were collected from the feces of the macaques
and directly from trees. The fecal samples were stored in plastic bags with infomation of
individuals if available. These fecal samples were washed on a fine sieve and the solid
insoluble-contents of the feces (hair, undigested animal and plant matter including the
seed) were dried overnight at room temperature. Based on the morphology of the
seeds, they were categorized into different types (species). A total of 20 such seed
samples were used for DNA analysis. Ten leaf samples from six species of ficus known
to occur in the region were also collected and identified based on their morphology.
These samples would be analyzed and used as controls for the seed samples.
3) Sex identification using monkey DNA
A. Isolation of DNA from the feces for sex identification
DNA was extracted from the ten samples using QIAamp DNA Stool Mini Kit according
to manufacturer’s instructions with minor modification.
B. PCR amplification
For sexing (with two positive controls)
We amplified two regions (ZFX and SRY) simultaneously. ZFX is a region on X
and Y-chromosome, and SRY is a region on only Y-chromosome. Consequently, male
samples yield two different size PCR products (445bp and 224bp), while female
samples lack one of them (we find only 445bp bands). PCR products were checked by
agarose gel electrophoresis, and then we can identify the sexes.
PCR was carried out in 10 μl reactions containing 5μl of AmpliTaq Gold master mix
(Applied Biosystems), 0.5 μM of forward and reverse primers of ZFX (F:
ATAATCACATGGAGAGCCACAAGCT , R:
GCACTTCTTTGGTATCTGAGAAAGT) and SRY (F:
CCCATGAACGCATTCATTGTGTGG, R: ATTTTAGCCTTCCGACGAGGTCGATA)
and 1μl DNA extract. After an initial incubation of 95 °C for 9 min, PCR amplification
was conducted for 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at
60 °C for 1 min, extension at 72 °C for 1 min and a final extension at 72 °C for 5 min.
4) Species identification using plant DNA
A. Isolation of DNA from seeds and leaves for species identification.
Plant DNA was extracted from 20 seeds collected from 12 fecal samples and from 12
leaf samples as control.
First, 10-15 grains and 5 × 5 mm pieces of leaves were ground. Then we extracted
DNA using DNeasy Plant Mini Kit according to manufacturer’s instructions.
For species identification of plants
We extracted DNA from the fig seeds in the feces and leaves of figs. We
amplified rbcL and internal transcribed spacer (ITS) regions (Kita and Ito 2000, Azuma
et al. 2010, Li et al. 2012). Those regions are two of the common markers for plant
DNA barcode system. RbcL was located in chloroplast genome and ITS was a nucleic
region. Only ITS region can distinguish the species well, but are difficult to amplify
because the copy number of nucleic DNA is quite smaller than chloroplast DNA. So, if
we succeed amplifying ITS region, we will sequence this region. If we succeed
amplifying only rbcL region, we will sequence this region. In the latter case, detailed
species identification may be difficult.
PCR was carried out in 15 μl reactions containing 0.75 U of Ex Taq enzymes
(Takara), 1μM of forward and reverse primers, 2mM of each dNTP, PCR Buffer and
1.5μl of DNA extracts. Primers for RbcL were followings; rbcL1F:
ATGTCACCACAAACAGAAAC and 724R: TCGCATGTACCTGCAGTAGC. Primers
for ITS were ITS-Y5: TAGAGGAAGGAGAAGTCGTAACAA and ITS-Y4:
CCCGCCTGACCTGGGGTCGC. After an initial incubation of 94 °C for 5 min, PCR
amplification was conducted for 35 cycles consisting of denaturation at 94 °C for 30 s,
annealing at 48 °C (rbcL) or 50 °C (ITS) for 30 min, extension at 72 °C for 1 min and a
final extension at 72 °C for 7 min.
After checking PCR products using agalose gel electrophoresis, the products
were cleaned up using ExoSap-IT, sequence reaction was conducted using BigDye
Terminator ver3.1, and then sequence run was performed in ABI 3130xL.
Sequence results were analyzed using MEGA ver 4.0.2 and ficus species were
identified by using BLAST in NCBI database. Yakushima has six species of ficus and
all of which have registered ITS sequences. Neighbor joining phylogenetic tree was
then constructed using MEGA ver 4.0.2.
RESULTS
1. Sex identification in macaques
DNA was extracted from fecal samples and a blood sample of ten macaques. We
performed agarose gel electrophoresis and analyzed them to identify the sex of
macaques. We compared the sex of macaques between the results obtained from direct
observation and DNA analysis. We tested this experiment by using markers (ZFX and
SRY regions). We observed the DNA bands. As a result; we identified the sex of
macaques and found out that five among ten samples were females. The result also
matched the direct observation of known individual by a hundred percent.
1 2 3 4 5 6 7 8 9 10 11 12 Marker
Figure 1: Agarose gel electrophoresis of DNA from Japanese macaques. (1), (4), (5) (7)
and (9) are male macaques. (2), (3), (6) female macaques.
1353bp
1078bp
872bp
603bp
1310bp
1353bp
1353bp
F: Female C: Control
M: Male and B: Blood
2. Identification of ficus species from macaque feces
Plant DNA was extracted from seeds collected from macaque feces to identify
the ficus species fed by them. We analyzed the sequences and constructed a neighbor
joining phylogenetic tree using the best matches to our sequences obtained from the
National Centre for Biotechnology Information (NCBI) nucleotide database by using
Basic Local Alignment Search Tool (BLAST). A series of registered internal transcriber
spacer (ITS) sequences of six ficus species were used as references. Figure 2 shows the
phylogenetic tree. As a result the genetic analysis shows that macaques fed mostly on
Ficus superba and F. thunbergii among the six ficus species. However, a clear
assignment of the seeds to either F. superba or F. thunbergii was not possible.
DISCUSSION
We made an attempt to study the feeding behavior of Yakushima macaques by
using molecular technique. Major advantage of non–invasive methods is that, we can
understand the given species without disturbing them. However we need long term
observations, to completely understand their feeding ecology.
Our first objective was to carry out molecular sexing of the individuals that we
had observed. Though, we can identify sex of the individual by visual observations, this
technique is very much useful in validating the collected data and in areas where
observations are difficult to carry out. Totally we collected nine fecal samples and a
blood sample. DNA was extracted followed by agarose gel electrophoresis. With the
help of amplified regions ZXF which is present on both X and Y-chromosome and SRY
is the region present only on Y-chromosome. From the collected samples, five of them
were from females and rest of them was from males. By running Agarose gel
electrophoresis, we can differentiate whether the given sample is from male or male.
Male samples yielded two different size PCR products (445bp and 224bp), while female
samples lack one of them (445bp bands).
Main objective of the study was to understand the macaques feeding behavior.
That can be easily understood by examining the feces of macaques. We collected seeds
from their feces and leaves of fig trees. The latter was used as a control. For DNA
sequencing, two amplified regions were used they are, rbcL and internal transcribed
spacer (ITS) regions (Kita and Ito 2000, Azuma et. al., 2010 and Li et. al., 2012). These
regions are the most common markers used for plant DNA barcode system. RbcL is
located in chloroplast genome and ITS is present in nucleic region. Later sequences
were analyzed using MEGA 4.0 software. The final sequence was compared with the
sequence obtained from the NCBI nucleotide database with the help of BLAST, which
has registered ITS sequences of many plant and animal species. We mainly looked for
six ficus species, which are widely distributed in Yakushima Island and monkeys
predominantly feed upon.
We found that Yakushima macaques mainly feed on Ficus superba and Ficus
thunbergii. Apart from ficus, we observed, monkeys eating mushrooms and insects
(mostly Beetles, sometimes other small insects). By using this little information, we
constructed phylogenetic tree which explains distance between each species sequence
from one another.
CONCLUSION
Monkeys are believed to be generalist feeders. To prove this we might need long
term observations on different individuals, in different seasons, in different altitudinal
ranges.
Non invasive techniques greatly help to understand certain valuable information
about species which can’t be obtained from simple observations. To understand
Yakushima macaques feeding ecology, long term observation is strongly recommended.
ACKNOWLEDGMENT
We would like to thank the following institutions and individuals for logistic,
technical and monetary support during the course of study. During the field course, Dr.
Sugiura, Mr. Suzumura, Dr. Takahashi and Dr. Sawada taught us about monkeys. And
during the genome course, Dr. Inoue, Mr. Adenyo and Mr. Sherif taught us about DNA
analysis. So they supported us in each course. Moreover, we are thankful to all people
who were involved in the preparation of the field and genome course; including Dr.
Kohshima and Wildlife Research Centre of Kyoto University.
REFERENCES
Agetsuma, N. 1995. Foraging strategies of Yakushima macaques (Macaca fuscata
yakui). International Journal of Primatology 16: 595-609
Azuma, H., Harrison, R.D., Nakamura, K. & Hui, Z. 2010. Molecular phylogenies of figs
and fig-pollinating wasps in the Ryukyu and Bonin (Ogasawara) islands, Japan.
Genes & Genetic Systems 85: 177-192.
Hill, D.A. 1997. Seasonal Variation in the Feeding Behavior and Diet of Japanese
Macaque (Macaca fuscata yakui) in Lowland Forest of Yakushima. United
Kingdom. American Journal of Primatology 43:305-322
Inoue, M. & Akenaka, O. 1993. Japanese macaque microsatellite PCR primers for
paternity testing. Primates 34: 37-45
Kita, Y. & Ito, M. 2000. Nuclear ribosomal ITS sequences and phylogeny in East
Asian Aconitum subgenusAconitum (Ranunculaceae), with special reference
to extensive polymorphism in individual plants, Plants systematics and
Evolution 225: 1-13.
Li. J.J., Liu, Y., Xin, X., Kim, T.S., Cabeza, E.A., Ren, J., Nielsen, R., Wrana, J.L. &
Zhang, Z. 2012. Evidence for Positive Selection on a Number of MicroRNA
Regulatory Interactions during Recent Human Evolution.
Maruhashi, T. 1980. Feeding behavior and the diet of the Japanese monkey (Macaca
fuscata yakui) on Yakushima Island, Japan. Primates 21: 141-160
Otani, T. & Shibata, E. 2000. Seed dispersal and predation by Yakushima macaques,
Macaca fuscata yakui, in a warm temperate forest of Yakushima Island,
southern Japan. Ecological Research 15: 133-144
Wong, C.M. 2012. Manual Laboratory DNA Extraction and PCR Amplification.
Universiti Malaysia Sabah.